Naive T-Cell Depletion to Prevent Chronic Graft-Versus-Host Disease.

PURPOSE: Graft-versus-host disease (GVHD) causes morbidity and mortality following allogeneic hematopoietic cell transplantation. Naive T cells (TN) cause severe GVHD in murine models. We evaluated chronic GVHD (cGVHD) and other outcomes in three phase II clinical trials of TN-depletion of peripheral blood stem-cell (PBSC) grafts. METHODS: One hundred thirty-eight patients with acute leukemia received TN-depleted PBSC from HLA-matched related or unrelated donors following conditioning with high- or intermediate-dose total-body irradiation and chemotherapy. GVHD prophylaxis was with tacrolimus, with or without methotrexate or mycophenolate mofetil. Subjects received CD34-selected PBSC and a defined dose of memory T cells depleted of TN. Median follow-up was 4 years. The primary outcome of the analysis of cumulative data from the three trials was cGVHD. RESULTS: cGVHD was very infrequent and mild (3-year cumulative incidence total, 7% [95% CI, 2 to 11]; moderate, 1% [95% CI, 0 to 2]; severe, 0%). Grade III and IV acute GVHD (aGVHD) occurred in 4% (95% CI, 1 to 8) and 0%, respectively. The cumulative incidence of grade II aGVHD, which was mostly stage 1 upper gastrointestinal GVHD, was 71% (95% CI, 64 to 79). Recipients of matched related donor and matched unrelated donor grafts had similar rates of grade III aGVHD (5% [95% CI, 0 to 9] and 4% [95% CI, 0 to 9]) and cGVHD (7% [95% CI, 2 to 13] and 6% [95% CI, 0 to 12]). Overall survival, cGVHD-free, relapse-free survival, relapse, and nonrelapse mortality were, respectively, 77% (95% CI, 71 to 85), 68% (95% CI, 61 to 76), 23% (95% CI, 16 to 30), and 8% (95% CI, 3 to 13) at 3 years. CONCLUSION: Depletion of TN from PBSC allografts results in very low incidences of severe acute and any cGVHD, without apparent excess risks of relapse or nonrelapse mortality, distinguishing this novel graft engineering strategy from other hematopoietic cell transplantation approaches.

Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel.

The ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34+ cord blood and bone-marrow-derived HSPCs. This culture system led to a 73-fold increase in long-term hematopoietic stem cell (LT-HSC) frequency, as demonstrated by limiting dilution assays, and the expanded HSPCs were capable of hematopoietic reconstitution for at least 24 weeks in immunocompromised mice. Both the zwitterionic characteristics of the hydrogel and the 3D format were important for HSPC self-renewal. Mechanistically, the impact of 3D zwitterionic hydrogel culture on mitigating HSPC differentiation and promoting self-renewal might result from an inhibition of excessive reactive oxygen species (ROS) production via suppression of O2-related metabolism. HSPC expansion using zwitterionic hydrogels has the potential to facilitate the clinical application of hematopoietic-stem-cell therapies.

Myeloablative Autologous Stem-Cell Transplantation for Severe Scleroderma.

BACKGROUND: Despite current therapies, diffuse cutaneous systemic sclerosis (scleroderma) often has a devastating outcome. We compared myeloablative CD34+ selected autologous hematopoietic stem-cell transplantation with immunosuppression by means of 12 monthly infusions of cyclophosphamide in patients with scleroderma. METHODS: We randomly assigned adults (18 to 69 years of age) with severe scleroderma to undergo myeloablative autologous stem-cell transplantation (36 participants) or to receive cyclophosphamide (39 participants). The primary end point was a global rank composite score comparing participants with each other on the basis of a hierarchy of disease features assessed at 54 months: death, event-free survival (survival without respiratory, renal, or cardiac failure), forced vital capacity, the score on the Disability Index of the Health Assessment Questionnaire, and the modified Rodnan skin score. RESULTS: In the intention-to-treat population, global rank composite scores at 54 months showed the superiority of transplantation (67% of 1404 pairwise comparisons favored transplantation and 33% favored cyclophosphamide, P=0.01). In the per-protocol population (participants who received a transplant or completed ≥9 doses of cyclophosphamide), the rate of event-free survival at 54 months was 79% in the transplantation group and 50% in the cyclophosphamide group (P=0.02). At 72 months, Kaplan-Meier estimates of event-free survival (74% vs. 47%) and overall survival (86% vs. 51%) also favored transplantation (P=0.03 and 0.02, respectively). A total of 9% of the participants in the transplantation group had initiated disease-modifying antirheumatic drugs (DMARDs) by 54 months, as compared with 44% of those in the cyclophosphamide group (P=0.001). Treatment-related mortality in the transplantation group was 3% at 54 months and 6% at 72 months, as compared with 0% in the cyclophosphamide group. CONCLUSIONS: Myeloablative autologous hematopoietic stem-cell transplantation achieved long-term benefits in patients with scleroderma, including improved event-free and overall survival, at a cost of increased expected toxicity. Rates of treatment-related death and post-transplantation use of DMARDs were lower than those in previous reports of nonmyeloablative transplantation. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institutes of Health; ClinicalTrials.gov number, NCT00114530 .).

Preclinical Optimization of a CD20-specific Chimeric Antigen Receptor Vector and Culture Conditions.

Chimeric antigen receptor (CAR)-based adoptive T-cell therapy is a highly promising treatment for lymphoid malignancies, and CD20 is an ideal target antigen. We previously developed a lentiviral construct encoding a third generation CD20-targeted CAR but identified several features that required additional optimization before clinical translation. We describe here several improvements, including replacement of the immunogenic murine antigen-binding moiety with a fully human domain, streamlining the transgene insert to enhance lentiviral titers, modifications to the extracellular IgG spacer that abrogate nonspecific activation resulting from binding to Fc receptors, and evaluation of CD28, 4-1BB, or CD28 and 4-1BB costimulatory domains. We also found that restimulation of CAR T cells with an irradiated CD20 cell line boosted cell growth, increased the fraction of CAR-expressing cells, and preserved in vivo function despite leading to a reduced capacity for cytokine secretion in vitro. We also found that cryopreservation of CAR T cells did not affect immunophenotype or in vivo antitumor activity compared with fresh cells. These optimization steps resulted in significant improvement in antitumor activity in mouse models, resulting in eradication of established systemic lymphoma tumors in 75% of mice with a single infusion of CAR T cells, and prolonged in vivo persistence of modified cells. These results provide the basis for clinical testing of a lentiviral construct encoding a fully human CD20-targeted CAR with CD28 and 4-1BB costimulatory domains and truncated CD19 (tCD19) transduction marker.

Durable Molecular Remissions in Chronic Lymphocytic Leukemia Treated With CD19-Specific Chimeric Antigen Receptor-Modified T Cells After Failure of Ibrutinib.

Purpose We evaluated the safety and feasibility of anti-CD19 chimeric antigen receptor-modified T (CAR-T) cell therapy in patients with chronic lymphocytic leukemia (CLL) who had previously received ibrutinib. Methods Twenty-four patients with CLL received lymphodepleting chemotherapy and anti-CD19 CAR-T cells at one of three dose levels (2 × 105, 2 × 106, or 2 × 107 CAR-T cells/kg). Nineteen patients experienced disease progression while receiving ibrutinib, three were ibrutinib intolerant, and two did not experience progression while receiving ibrutinib. Six patients were venetoclax refractory, and 23 had a complex karyotype and/or 17p deletion. Results Four weeks after CAR-T cell infusion, the overall response rate (complete response [CR] and/or partial response [PR]) by International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria was 71% (17 of 24). Twenty patients (83%) developed cytokine release syndrome, and eight (33%) developed neurotoxicity, which was reversible in all but one patient with a fatal outcome. Twenty of 24 patients received cyclophosphamide and fludarabine lymphodepletion and CD19 CAR-T cells at or below the maximum tolerated dose (≤ 2 × 106 CAR-T cells/kg). In 19 of these patients who were restaged, the overall response rate by IWCLL imaging criteria 4 weeks after infusion was 74% (CR, 4/19, 21%; PR, 10/19, 53%), and 15/17 patients (88%) with marrow disease before CAR-T cells had no disease by flow cytometry after CAR-T cells. Twelve of these patients underwent deep IGH sequencing, and seven (58%) had no malignant IGH sequences detected in marrow. Absence of the malignant IGH clone in marrow of patients with CLL who responded by IWCLL criteria was associated with 100% progression-free survival and overall survival (median 6.6 months follow-up) after CAR-T cell immunotherapy. The progression-free survival was similar in patients with lymph node PR or CR by IWCLL criteria. Conclusion CD19 CAR-T cells are highly effective in high-risk patients with CLL after they experience treatment failure with ibrutinib therapy.

Manufacture of Autologous CD34+ Selected Grafts in the NIAID-Sponsored HALT-MS and SCOT Multicenter Clinical Trials for Autoimmune Diseases.

To ensure comparable grafts for autologous hematopoietic cell transplantation (HCT) in the National Institute of Allergy and Infectious Diseases-sponsored Investigational New Drug protocols for multiple sclerosis (HALT-MS) and systemic sclerosis (SCOT), a Drug Master File approach to control manufacture was implemented, including a common Master Production Batch Record and site-specific standard operating procedures with "Critical Elements." We assessed comparability of flow cytometry and controlled rate cryopreservation among sites and stability of cryopreserved grafts using hematopoietic progenitor cells (HPCs) from healthy donors. Hematopoietic Progenitor Cells, Apheresis-CD34+ Enriched, for Autologous Use (Auto-CD34+HPC) graft specifications included ≥70% viable CD34+ cells before cryopreservation. For the 2 protocols, 110 apheresis collections were performed; 121 lots of Auto-CD34+HPC were cryopreserved, and 107 of these (88.4%) met release criteria. Grafts were infused at a median of 25 days (range, 17 to 68) post-apheresis for HALT-MS (n = 24), and 25 days (range, 14 to 78) for SCOT (n = 33). Subjects received precryopreservation doses of a median 5.1 × 106 viable CD34+ cells/kg (range, 3.9 to 12.8) for HALT-MS and 5.6 × 106 viable CD34+ cells/kg (range, 2.6 to 10.2) for SCOT. Recovery of granulocytes occurred at a median of 11 days (range, 9 to 15) post-HCT for HALT-MS and 10 days (range, 8 to 12) for SCOT, independent of CD34+ cell dose. Subjects received their last platelet transfusion at a median of 9 days (range, 6 to 16) for HALT-MS and 8 days (range, 6 to 23) for SCOT; higher CD34+/kg doses were associated with faster platelet recovery. Stability testing of cryopreserved healthy donor CD34+ HPCs over 6 months of vapor phase liquid nitrogen storage demonstrated consistent 69% to 73% recovery of viable CD34+ cells. Manufacturing of Auto-CD34+HPC for the HALT-MS and SCOT protocols was comparable across all sites and supportive for timely recovery of granulocytes and platelets.

Notch-Expanded Murine Hematopoietic Stem and Progenitor Cells Mitigate Death from Lethal Radiation and Convey Immune Tolerance in Mismatched Recipients.

The hematopoietic syndrome of acute radiation syndrome (h-ARS) is characterized by severe bone marrow aplasia, resulting in a significant risk for bleeding, infections, and death. To date, clinical management of h-ARS is limited to supportive care dictated by the level of radiation exposure, with a high incidence of mortality in those exposed to high radiation doses. The ideal therapeutic agent would be an immediately available, easily distributable single-agent therapy capable of rapid in vivo hematopoietic reconstitution until recovery of autologous hematopoiesis occurs. Using a murine model of h-ARS, we herein demonstrate that infusion of ex vivo expanded murine hematopoietic stem and progenitor cells (HSPCs) into major histocompatibility complex mismatched recipient mice exposed to a lethal dose of ionizing radiation (IR) led to rapid myeloid recovery and improved survival. Survival benefit was significant in a dose-dependent manner even when infusion of the expanded cell therapy was delayed 3 days after lethal IR exposure. Most surviving mice (80%) demonstrated long-term in vivo persistence of donor T cells at low levels, and none had evidence of graft versus host disease. Furthermore, survival of donor-derived skin grafts was significantly prolonged in recipients rescued from h-ARS by infusion of the mismatched expanded cell product. These findings provide evidence that ex vivo expanded mismatched HSPCs can provide rapid, high-level hematopoietic reconstitution, mitigate IR-induced mortality, and convey donor-specific immune tolerance in a murine h-ARS model. Stem Cells Translational Medicine 2017;6:566-575.

Real Time Immunophenotyping of Leukocyte Subsets Early after Double Cord Blood Transplantation Predicts Graft Function.

Cord blood transplantation (CBT) recipients are at increased risk for delayed engraftment and primary graft failure, complications that are often indistinguishable early post-transplantation. Current assays fail to accurately identify recipients with slow hematopoietic recovery and distinguish them from those with pending graft failure. To address this, we prospectively examined the kinetics of immune cell subset recovery in the peripheral blood of 39 patients on days +7 and +14 after double-unit CBT (dCBT) by multiparametric flow cytometry analysis, which we term real-time immunophenotyping (RTIP). RTIP analysis at day +14 revealed distinctive patterns of reconstitution and, importantly, identified patients with slow hematopoietic recovery who went on to engraft. Strikingly, higher absolute numbers of circulating monocytes and natural killer cells at day +14 were predictive of engraftment, but only the absolute number of circulating monocytes was significantly correlated with time to engraftment. This is the first evidence that RTIP on patient peripheral blood mononuclear cells early after dCBT is technically feasible and can be used as a "signature" for predicting the kinetics of hematopoietic recovery. Furthermore, RTIP is a time- and cost-efficient methodology that has the potential to become a clinically feasible diagnostic tool to guide therapeutic interventions in high-risk patients; therefore, its utility should be evaluated in a large cohort of patients.

Semi-automated closed system manufacturing of lentivirus gene-modified haematopoietic stem cells for gene therapy.

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. However, current state of the art requires sophisticated ex vivo gene transfer in a dedicated Good Manufacturing Practices facility, limiting availability. An automated process would improve the availability and standardized manufacture of HSC gene therapy. Here, we develop a novel program for semi-automated cell isolation and culture equipment to permit complete benchtop generation of gene-modified CD34+ blood cell products for transplantation. These cell products meet current manufacturing quality standards for both mobilized leukapheresis and bone marrow, and reconstitute human haematopoiesis in immunocompromised mice. Importantly, nonhuman primate autologous gene-modified CD34+ cell products are capable of stable, polyclonal multilineage reconstitution with follow-up of more than 1 year. These data demonstrate proof of concept for point-of-care delivery of HSC gene therapy. Given the many target diseases for gene therapy, there is enormous potential for this approach to treat patients on a global scale.

Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells.

CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4(+)/CD8(+) ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin's lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The CR rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR; n = 11). Cy/Flu minimized the effects of an immune response to the murine single-chain variable fragment component of the CAR, which limited CAR-T cell expansion and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥3 neurotoxicity were observed in 13 and 28% of all patients, respectively. Serum biomarkers, one day after CAR-T cell infusion, correlated with subsequent sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4(+)/CD8(+) ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of lymphodepletion that improved disease response and overall and progression-free survival.

CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients.

BACKGROUND: T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR-T cell products were prepared from unselected T cells. METHODS: We conducted a clinical trial to evaluate CD19 CAR-T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS: The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR-T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR-T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell-mediated anti-CAR transgene product immune responses developed after CAR-T cell infusion in some patients, limited CAR-T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR-T cell persistence and disease-free survival. CONCLUSION: Immunotherapy with a CAR-T cell product of defined composition enabled identification of factors that correlated with CAR-T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR-T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION: ClinicalTrials.gov NCT01865617. FUNDING: R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.

A Distributed Network for Intensive Longitudinal Monitoring in Metastatic Triple-Negative Breast Cancer.

Accelerating cancer research is expected to require new types of clinical trials. This report describes the Intensive Trial of OMics in Cancer (ITOMIC) and a participant with triple-negative breast cancer metastatic to bone, who had markedly elevated circulating tumor cells (CTCs) that were monitored 48 times over 9 months. A total of 32 researchers from 14 institutions were engaged in the patient's evaluation; 20 researchers had no prior involvement in patient care and 18 were recruited specifically for this patient. Whole-exome sequencing of 3 bone marrow samples demonstrated a novel ROS1 variant that was estimated to be present in most or all tumor cells. After an initial response to cisplatin, a hypothesis of crizotinib sensitivity was disproven. Leukapheresis followed by partial CTC enrichment allowed for the development of a differential high-throughput drug screen and demonstrated sensitivity to investigational BH3-mimetic inhibitors of BCL-2 that could not be tested in the patient because requests to the pharmaceutical sponsors were denied. The number and size of CTC clusters correlated with clinical status and eventually death. Focusing the expertise of a distributed network of investigators on an intensively monitored patient with cancer can generate high-resolution views of the natural history of cancer and suggest new opportunities for therapy. Optimization requires access to investigational drugs.

Outcomes of acute leukemia patients transplanted with naive T cell-depleted stem cell grafts.

BACKGROUND: Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HCT). In mice, naive T cells (TN) cause more severe GVHD than memory T cells (TM). We hypothesized that selective depletion of TN from human allogeneic peripheral blood stem cell (PBSC) grafts would reduce GVHD and provide sufficient numbers of hematopoietic stem cells and TM to permit hematopoietic engraftment and the transfer of pathogen-specific T cells from donor to recipient, respectively. METHODS: In a single-arm clinical trial, we transplanted 35 patients with high-risk leukemia with TN-depleted PBSC grafts following conditioning with total body irradiation, thiotepa, and fludarabine. GVHD prophylactic management was with tacrolimus immunosuppression alone. Subjects received CD34-selected PBSCs and a defined dose of TM purged of CD45RA+ TN. Primary and secondary objectives included engraftment, acute and chronic GVHD, and immune reconstitution. RESULTS: All recipients of TN-depleted PBSCs engrafted. The incidence of acute GVHD was not reduced; however, GVHD in these patients was universally corticosteroid responsive. Chronic GVHD was remarkably infrequent (9%; median follow-up 932 days) compared with historical rates of approximately 50% with T cell-replete grafts. TM in the graft resulted in rapid T cell recovery and transfer of protective virus-specific immunity. Excessive rates of infection or relapse did not occur and overall survival was 78% at 2 years. CONCLUSION: Depletion of TN from stem cell allografts reduces the incidence of chronic GVHD, while preserving the transfer of functional T cell memory. TRIAL REGISTRATION: ClinicalTrials.gov (NCT 00914940).

Association of fludarabine pharmacokinetic/dynamic biomarkers with donor chimerism in nonmyeloablative HCT recipients.

PURPOSE: Fludarabine monophosphate (fludarabine) is an integral component of many reduced-intensity conditioning regimens for hematopoietic cell transplantation (HCT). Fludarabine's metabolite, 9-ß-D-arabinofuranosyl-2-fluoroadenine (F-ara-A), undergoes cellular uptake and activation to form the active cytotoxic metabolite fludarabine triphosphate (F-ara-ATP), which inhibits cellular DNA synthesis in CD4(+) and CD8(+) cells. In this study, we evaluated whether fludarabine-based pharmacologic biomarkers were associated with clinical outcomes in HCT recipients. METHODS: Participants with hematologic diseases were conditioned with fludarabine and low-dose total body irradiation (TBI) followed by allogeneic HCT and post-grafting immunosuppression. After fludarabine administration, we evaluated pharmacological biomarkers for fludarabine-F-ara-A area under the curve (AUC) and the ratio of circulating CD4(+) and CD8(+) cells (CD4(+)/CD8(+) ratio) after fludarabine administration-in 102 patients; F-ara-ATP accumulation rate in enriched CD4(+) and CD8(+) cells was evaluated in 36 and 34 patients, respectively. RESULTS: Interpatient variability in the pharmacological biomarkers was high, ranging from 3.7-fold (F-ara-A AUC) to 39-fold (F-ara-ATP in CD8(+) cells). Circulating CD8(+) cells were more sensitive to fludarabine administration. A population pharmacokinetic-based sampling schedule successfully allowed for estimation of F-ara-A AUC in this outpatient population. There was a poor correlation between the F-ara-AUC and the F-ara-ATP accumulation rate in CD4(+) (R (2) = 0.01) and CD8(+) cells (R (2) = 0.00). No associations were seen between the four biomarkers and clinical outcomes (day +28 donor T cell chimerism, acute graft-versus-host disease (GVHD), neutrophil nadirs, cytomegalovirus reactivation, chronic GVHD, relapse, non-relapse mortality, or overall mortality). CONCLUSIONS: Considerable interpatient variability exists in pharmacokinetic and fludarabine-based biomarkers, but these biomarkers are not associated with clinical outcomes in fludarabine/TBI-conditioned patients.

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia.

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.

MIR144 and MIR451 regulate human erythropoiesis via RAB14.

Expression levels of MIR144 and MIR451 increase during erythropoiesis, a pattern that is conserved from zebrafish to humans. As these two miRs are expressed from the same polycistronic transcript, we manipulated MIR144 and MIR451 in human erythroid cells individually and together to investigate their effects on human erythropoiesis. Inhibition of endogenous human MIR451 resulted in decreased numbers of erythroid (CD71(hi) CD235a(hi) CD34(-) ) cells, consistent with prior studies in zebrafish and mice. In addition, inhibition of MIR144 impaired human erythroid differentiation, unlike in zebrafish and mouse studies where the functional effect of MIR144 on erythropoiesis was minimal. In this study, we found RAB14 is a direct target of both MIR144 and MIR451. As MIR144 and MIR451 expression increased during human erythropoiesis, RAB14 protein expression decreased. Enforced RAB14 expression phenocopied the effect of MIR144 and/or MIR451 depletion, whereas shRNA-mediated RAB14 knockdown protected cells from MIR144 and/or MIR451 depletion-mediated erythropoietic inhibition. RAB14 knockdown increased the frequency and number of erythroid cells, increased ß-haemoglobin expression, and decreased CBFA2T3 expression during human erythropoiesis. In summary, we utilized MIR144 and MIR451 to identify RAB14 as a novel physiological inhibitor of human erythropoiesis.

Tetramer guided, cell sorter assisted production of clinical grade autologous NY-ESO-1 specific CD8(+) T cells.

BACKGROUND: Adoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL). METHODS: CTL generation was evaluated in 6 patients with NY-ESO-1 positive sarcomas, under clinical manufacturing conditions and characterized for phenotypic and functional properties. RESULTS: Following in vitro stimulation, T cells stained with NY-ESO-1 tetramer were enriched from frequencies as low as 0.4% to >90% after single pass through a clinical grade sorter. NY-ESO-1 specific T cells were generated from all 6 patients. The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8(+), tetramer(+) T cells with a memory phenotype that recognized endogenous NY-ESO-1. CONCLUSION: This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy. This approach, when used to target more broadly expressed tumor antigens such as WT-1 and additional Cancer-Testis antigens will enhance the scope and feasibility of adoptive T cell therapy.

Engineering human peripheral blood stem cell grafts that are depleted of naïve T cells and retain functional pathogen-specific memory T cells.

Graft-versus-host disease (GVHD) is a frequent major complication of allogeneic hematopoietic cell transplantation (HCT). Approaches that selectively deplete T cells that cause GVHD from allogeneic stem cell grafts and preserve T cells specific for pathogens may improve HCT outcomes. It has been hypothesized that the majority of T cells that can cause GVHD reside within the naïve T cell (TN) subset, and previous studies performed in mouse models and with human cells in vitro support this hypothesis. As a prelude to translating these findings to the clinic, we developed and evaluated a novel 2-step clinically compliant procedure for manipulating peripheral blood stem cells (PBSC) to remove TN, preserve CD34(+) hematopoietic stem cells, and provide for a fixed dose of memory T cells (TM) that includes T cells with specificity for common opportunistic pathogens encountered after HCT. Our studies demonstrate effective and reproducible performance of the immunomagnetic cell selection procedure for depleting TN. Moreover, after cell processing, the CD45RA-depleted PBSC products are enriched for CD4(+) and CD8(+) TM with a central memory phenotype and contain TM cells that are capable of proliferating and producing effector cytokines in response to opportunistic pathogens.

Developmental fate and cellular maturity encoded in human regulatory DNA landscapes.

Cellular-state information between generations of developing cells may be propagated via regulatory regions. We report consistent patterns of gain and loss of DNase I-hypersensitive sites (DHSs) as cells progress from embryonic stem cells (ESCs) to terminal fates. DHS patterns alone convey rich information about cell fate and lineage relationships distinct from information conveyed by gene expression. Developing cells share a proportion of their DHS landscapes with ESCs; that proportion decreases continuously in each cell type as differentiation progresses, providing a quantitative benchmark of developmental maturity. Developmentally stable DHSs densely encode binding sites for transcription factors involved in autoregulatory feedback circuits. In contrast to normal cells, cancer cells extensively reactivate silenced ESC DHSs and those from developmental programs external to the cell lineage from which the malignancy derives. Our results point to changes in regulatory DNA landscapes as quantitative indicators of cell-fate transitions, lineage relationships, and dysfunction.

Correlation of infused CD3+CD8+ cells with single-donor dominance after double-unit cord blood transplantation.

Single-donor dominance is observed in the majority of patients following double-unit cord blood transplantation (dCBT); however, the biological basis for this outcome is poorly understood. To investigate the possible influence of specific cell lineages on dominance in dCBT, flow cytometry assessment for CD34(+), CD14(+), CD20(+), CD3(-)CD56(+), CD3(+)CD56(+) (natural killer), and T cell subsets (CD4(+), CD8(+), memory, naïve, and regulatory) was performed on individual units. Subsets were calculated as infused viable cells per kilogram of recipient actual weight. Sixty patients who underwent dCBT were included in the final analysis. Higher CD3(+) cell dose was statistically concordant with the dominant unit in 72% of cases (P = .0006). Further T cell subset analyses showed that dominance was correlated more with the naive CD8(+) cell subset (71% concordance; P = .009) than with the naive CD4(+) cell subset (61% concordance; P = .19). These data indicate that a greater total CD3(+) cell dose, particularly of naïve CD3(+)CD8(+) T cells, may play an important role in determining single-donor dominance after dCBT.